A long-term objective of this proposal is to elucidate the structural and dynamic properties of unfolded proteins and transient intermediate states populated during the initial stages of folding. Molecular site-resolved information on local and long-range structural propensities in these states is critical for understanding how the native structure of a protein, and ultimately its function, are encoded in the amino acid sequence. We will study the conformational and dynamic properties of solvent-denatured staphylococcal nuclease and cytochrome c, as well as the intrinsically disordered apocytochrome c, using solution NMR, including paramagnetic relaxation enhancement and gradient diffusion methods. Structure formation during early stages of folding of these proteins will be monitored by combining H/D exchange with ultra-fast mixing and NMR analysis. Critical residues involved in stabilizing non-random structural features involving long-range and/or local interactions will be identified by measuring the effects of mutations on the hydrodynamic dimensions and relaxation profiles. The lessons learned and approaches developed will benefit a new direction of our research aimed at understanding the conformational properties, dynamics and functional significance of intrinsically disordered regions in modular multidomain proteins. Intrinsic disorder is especially common in cell-signaling and cancer-associated proteins where individual structured domains are often connected via long flexible linkers. We will perform detailed studies of the structure, dynamics and binding properties of Na+/H+ exchanger regulatory factor 1 (NHERF1), a signaling adaptor comprised of two globular domains belonging to the large PDZ fold family, a C-terminal ezrin-binding motif and long disordered regions. A wide array of biophysical approaches, including structural and dynamic NMR techniques, thermodynamic analysis of mutant proteins, kinetic studies using advanced rapid mixing techniques and computational methods, will be applied. The findings will provide new insight into the role of intrinsically disordered regions in regulating the balance between intramolecular (autoinhibitory) domain-domain interactions and intermolecular ligand interactions. Ultimately, our work will lead to a better understanding of the mechanisms by which cell-signaling proteins, such as NHERF1, function as molecular switches. Together with our analysis of the structural tendencies in intrinsically disordered and chemically unfolded forms of globular proteins, the findings will shed new light on the sequence characteristics responsible for the subtle balance between order and disorder in polypeptide chains.